The NS1 protein of influenza A virus is a master regulator of the type I IFN-mediated antiviral response induced during viral infection. An important step of this response that is inhibited by this protein is the activation of transcription factors involved in IFN synthesis by the cytoplasmic RNA sensor of viral infection RIG-I. In collaboration with Dr. Jung, overall PI of this U19 proposal, we have found that the NS1 prevents activation of RIG-I by inhibiting its ubiquitination. Specifically, the NS1 binds to and inhibit TRIM25, a cellular ubiquitin ligase that ubiquitinates RIG-I, a process required for RIG-I downstream signaling. Our preliminary evidence also indicates that a mutant influenza A virus expressing an NS1 defective in its ability to bind and inhibit TRIM25 is attenuated and induces more IFN than wild type virus. In our proposed project, we will now investigate in detail the mechanism of inhibition of TRIM25 by the NS1 protein and the functional consequences of this inhibition on the host antiviral response and in the pathogenicity of influenza A virus. Since the NS1 protein has a complex set of nuclear import and export signals regulating its intracellular localization, we will also investigate the relationship between NS1 localization and function, and how NS1 localization is regulated during viral infection. Trafficking studies of NS1 in the cell will be performed with the help of the Imaging Core. These studies will provide us with a better understanding of the suppression of type I IFN induction by influenza virus. Our project will be complemented by Projects 1 and 3, that study the opposite side of the virus-host IFN response, i.e. induction of RIG-I activation; Project 2, that will provide us with structural clues on how RIG-I can be activated/inhibited; and Project 4, that study the impact of a different RNA virus, hepatitis C virus, in the host response, allowing us to compare different mechanisms used by two viruses to evade innate immunity.